SEMINARS IN LIVER DISEASE-VOL.

10, NO. 4, 1990

Role of Cytochromes P450 in Drug Metabolism and Hepatotoxicity

Over the last decade there has been an explosion in knowledge about a large group of liver enzymes collectively termed "the cytochromes P450." It has become virtually impossible for a clinician to avoid the mention of this enzyme system when reading about such diverse topics as prostaglandins, bile acids, or carcinogenesis. The purpose of this article is to acquaint clinicians with some of the recent discoveries concerning the cytochrome P450 system as they pertain to drug metabolism and hepatotoxicity. In order to understand the importance of these recent developments, it is necessary to review some basic principles about how drugs are handled by the body.

BASIC PRINCIPLES OF DRUG METABOLISM Most drugs that are administered to patients are fat soluble, or "lipophilic," to some degree. Solubility in fat is an essential property because the oral absorption of most drugs appears to involve passive diffusion through the apical, lipid, membranes of the enterocytes lining the gut. Drugs lacking lipophilicity are usually poorly absorbed by the oral route and therefore pass unaltered from the body in the stool. Once absorbed, lipophilic drugs circulate in blood largely bound to plasma proteins or are sequestered into fat and are therefore not readily excreted by the kidney into urine. Thus, the efficient elimination of lipophilic drugs relies on their conversion within the body to more water-soluble metabolites. Although many tissues and organs in the body appear capable of generating water-soluble metabolites from at least some drugs, the major organ with this function is the liver. The liver is uniquely suited to metabolize lipophilic drugs. This is because the endothelium lining the sinusoidal blood spaces contains transcellular pores, or "fenestrations," which allow passage of most blood proteins. No other organ contains vascular endothelial cells with

From the Department of Medicine, Division of Gastroenterology, University of Michigan Medical Center, Ann Arbor, Michigan. Reprint requests: Dr. Watkins, Department of Medicine, Division of Gastroenterology, University of Michigan Medical Center, 1150 W. Medical Center Drive, Ann Arbor, MI 48109.

such wide open "holes." As shown in Figure 1, blood proteins can therefore passively diffuse from the sinusoid into the space of Disse. As a result, drugs bound to protein are able to come into direct contact with the hepatocyte plasma membrane and can thereby diffuse or be actively transported into the hepatocyte. Once inside the hepatocyte, drugs can be converted into more water-soluble metabolites, which are often excreted back into the space of Disse. The metabolites will then enter the sinusoids and systemic circulation dissolved in the plasma water and thus be more readily excretable by the kidney. Alternatively, metabolites generated within the hepatocyte can be sorted to the canalicular membrane and be excreted in bile. Water-soluble metabolites in bile are generally poorly absorbed from the gastrointestinal tract and will largely be excreted in the stool. In summary, the liver plays a critical role in converting many drugs to water-soluble metabolites. This process is often rate limiting in the elimination of drugs from the body.

BRIEF HISTORY OF DRUG METABOLISM RESEARCH To put our current understanding of drug metabolites into appropriate perspective, it is important to understand the chronology of discoveries in the field. A more complete review of this topic, with appropriate literature citations, is available. ' In the 1940s, techniques were developed that were capable of isolating relatively pure subcellular fractions from whole liver tissue. The rough and smooth endoplasmic reticulum cannot be isolated as intact structures but can be selectively precipitated from hepatocyte lysate as a pellet containing tiny broken membrane vesicles. These fractions are reddish brown and are termed "microsomes." In the early 1950s, it was discovered that when reduced nicotinamide-adenine dinucleotide phosphate (NADPH) was added to liver microsomes, they were capable of catalyzing the majority of drug transformations known to occur in the liver in vivo. Because other subcellular fractions appeared to have little activity in this regard, the endoplasmic reticulum was identified as the major location for the drug metabolizing enzymes. During the next decade, investigators analyzed the metabolism of literally thousands of compounds by liver

Copyright 0 1990 by Thierne Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.

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PAUL B. WATKINS, M.D.

FIG. 1. How the liver handles drugs. Lipophilic drugs (white circles) exist in blood largely bound to proteins (white squares). Protein-bound drugs can enter the space of Disse through fenestrations in the sinusoidal endothelium. Drugs can then enter the hepatocyte and be converted to more water-soluble metabolites (dark circles) by enzymes present in the endoplasmic reticulum. The metabolites are then either regurgitated back into the sinusoidal blood or are sorted to the biliary canaliculus.

microsomes. Although the microsomes were capable of metabolizing a vast array of structurally diverse substances, the majority of reactions catalyzed were of two types: oxidations or conjugations. In most cases, oxidations involved the insertion into the drug of one atom of oxygen (derived from molecular oxygen) usually forming a hydroxyl group (i.e. hydroxylation). In conjugation reactions, a drug or drug metabolite was covalently attached to a polar (water-soluble) ligand such as sulfate or glucuronic acid. These two types of reactions were termed, respectively, "phase I" and phase 11" reactions because many drugs appeared to first require oxidation before they could undergo conjugation. In most instances, the sulfate or glucuronic acid is bound at the site of a hydroxyl group created by the phase I reaction. The bulk of early research within the field of drug metabolism was on phase I and not phase I1 enzymes. This was because studies in rats had shown that the liver's ability to perform phase I metabolism on some drugs could be dramatically increased ("induced") by pretreating the animals with phenobarbital or with 3-methylcholanthrene, a polycyclic aromatic hydrocarbon. Liver microsomes prepared from induced animals therefore contained a large amount of phase I catalytic activity, which could be easily studied. Moreover, because the microsomes from induced animals were noted to be red, initial attempts to purify enzymes from microsomes centered on isolating the red pigment they contained. This red pigment was found to be critical for phase I drug metabolizing activity in the microsomes. In contrast, phase 11 enzymes are colorless and often lose catalytic activity during purification. For this simple reason, the study of phase I1 enzymes has generally lagged behind that of the P450s. By the early 1960s, characterization of the red pigment in liver microsomes indicated that it was similar to heme proteins ("cytochromes") known to be present in mitochondria. The microsomal pigment bound oxygen

10, NUMBER 4, 1990

but (as is the case with another heme protein, hemoglobin), the oxygen could be displaced by carbon monoxide. In the presence of carbon monoxide, this pigment intensely absorbed light with wavelength of 4%) nanometers, resulting in the term "cytochrome pigment 450n, or "cytochrome P450. " (In keeping with a recent rec~mmendation,~ cytochrome P450 will be referred to as P450 in this review). P450 was shown to be critical for phase I metabolism because the oxidative reactions were completely inhibited when carbon monoxide was bubbled though liver microsomes. Moreover, the catalytic activity could be completely restored when the carbon monoxide was removed from the microsomes; this could be accomplished by exposing the microsomes to intense light with a wavelength of 450 nm. By the early 1970s, a variety of evidence suggested that the red pigment present in the microsomes consisted of multiple enzymes. First, it seemed inconceivable that a single enzyme could be capable of acting on all the structurally diverse substances that had been shown to undergo phase I metabolism in the microsomes. Second, when liver microsomes prepared from phenobarbital and 3-methylcholanthrene pretreated rats were compared, the ability to oxidize a battery of model substrates was not identical. Moreover, liver microsomes prepared from rats pretreated with 3-methylcholanthrene maximally absorbed light with a wavelength of 448 and not 450 nm. This led to the idea that two general types of P450 existed in microsomes, those inducible by phenobarbital (P450) and those inducible by 3-methylcholanthrene (P448). However, as attempts were made to purify P450 and P448, it was soon appreciated that many different P450s are in fact present in liver microsomes.' At current count over 20 different P450s have been identified in human liver and it seems likely that as many as 50 to 100 distinct P450 proteins may yet be discovered. The liver's ability to perform oxidative metabolism on a wide array of drugs results from multiple distinct P450s present in the endoplasmic reticulum of the hepatocyte.

GENETICS OF P450s Although it initially seemed likely that all P450s would be closely related in structure, this turned out not to be the case. Amino acid sequences determined directly from purified P450s, or deduced from the corresponding genes, have revealed marked heterogeneity, with some forms of P450 sharing less than 15% homology even within the same specie^.^ Each human P450 protein identified to date appears to reflect expression of a unique gene, and the P450 genes are distributed among at least several different chromosomes.4 The regulation of P450s has turned out to be very complex. In rats, a subset of P450 proteins is selectively induced by phenobarbital and others are selectively induced by 3-methy l c h ~ l a n t h r e n e ,providing ~,~ the molecular basis for early studies on P450 and P448 induction. However, some P450s are inducible by other types of compound^'^^ and still other P450s appear to be constitutively expressed The molecular biology and regulaand unindu~ib1e.~-'*

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SEMINARS IN LIVER DISEASE-VOLUME

tion of the P450s have been the subject of several recent reviews. I 3 - l 5 Despite marked differences in structure, regulation, and chromosomal location, there is significant homology between all P450s in a few regions of the protein^.'.^ Because of this, it is generally accepted that the mammalian P450 genes evolved from a common precursor gene well over 1 billion years ago (reviewed by Nebert The P450 genes have therefore been referred et a116-18). to as a "supergene fahily."' In an attempt to unify the nomenclature within the field of P450 research, a convention was recently adopted of grouping P450s that share greater than approximately 40% amino acid sequence homology into families.' The cut-off figure of 40% homology was arrived at somewhat arbitrarily based on P450 protein sequences known at the time the nomenclature was proposed.' At least 10 distinct P450 gene families have been identified to date.4 Many of these enzymes have been shown to be involved in physiologic processes, such as steroid hormone and prostaglandin b i o ~ y n t h e s i s .The ~ human P450s that have been identified as being important in drug metabolism belong to gene families I, 11, or 111. Table 1 lists some clinically important P450 proteins along with drugs and other xenobiotics that appear to characteristically induce them. It should be remembered that the study of inducible human liver P450s is in its infancy; the first report of selective induction of human hepatic P450 proteins by drugs appeared in 1985.19The confident identification of inducers in patients has been difficult because it is usually impossible in clinical studies to control all the variables that might influence P450 activities, such as diet and environmental exposures. Table 1 has therefore been constructed from data obtained from a variety of different types of studies. Some have quantitated individual P450 proteins or characteristic catalytic activities in liver

TABLE 1. Some Human Hepatic P450s and Their Probable Inducers Gene

Protc,ins

IA2

HLd," P450PA"

IIC*

P450mp,lh' P450meph" P450-8,-" HM-3"' P450DBJ' P450 buf l l h J HLj," P450ALCh'

IID* lIEl IIIA*

HLp," P450NFX1 HFLa,*.lh' H L P ~ , HLp3?[ ~' HPCN I , HPCN3" P450hA7,1M HM- I , HM-21h'

'"

Probable Ir7duc.rrs

Cigarette smoke'i '1 " " 1"' Charcoal broiled foods" " None identified None identified Ethanol" " Isoniazid" ~if~~~i~'x.1'1 Dexamethasone"' " " "' Cortisol'" Antiseizure medication'' (phenytoin, phenobarbital, carbamazepine) Phenylbuta~one'~ S~lfinpyrazone'~ Carbamazepine"

microsomes prepared from patients undergoing liver surgery and whose medication histories are known."-" Such studies usually seek to correlate findings with data obtained on induction in animal models. A second type of study involves induction of individual P450 proteins and catalytic activity in cultured human hepatocytes or hepatoma cell line^.'^-^^ Finally, the effect of treating patients with various suspected inducers has been assessed by studying the in vivo metabolism of model compounds that appear to be selectively acted on by individual p450s31-39 (see "How P450s Metabolize Drugs" later). Although each of these approaches has potential pitfalls, the aggregate data obtained to date strongly support selective induction of human liver P450s by medications that are in common clinical use. The P450I family appears to contain just two genes in man, only one of which (P450IA2) appears to be consistently expressed in human liver. In animals, these enzymes are selectively inducible by 3-methylcholanthrene and they therefore constitute what was previously termed "P448." The available evidence suggests that the human liver P-450IA2 enzyme is induced by cigarette smoking and consumption of charcoal-broiled foods. P-45011 is the largest family of human P450s identified to date. This family has been divided into subfamilies which share > 67% amino acid sequence homol~ g yAs . ~shown in Table 1, there are differences between P-45011 gene family members in terms of response to inducers. For example, the human P-450IIE subfamily member (P450IIE1) is inducible by ethanol and also appears to be inducible by isoniazid. This enzyme does not appear to be inducible by phenobarbital in patients. On the other hand, some members of the P-450IIC and IID subfamilies appear to be constitutively expressed in human liver because no inducers of these enzymes have been identified. The P-450111 gene family contains at least four very closely related genes (not listed in Table 1 ) that have been grouped into the subfamily "P450IIIA." These are major enzymes accounting for up to 25% of the total CObinding protein present in human liver microsomes. At least some of these enzymes are inducible by glucocorticoids, antiseizure medications, and macrolide antibiotics. 19.21.28.3YIt has recently been appreciated that expression of individual P450III genes is developmentally regulated. The major P450 present in fetal liver belongs to the P450III family (HFLa)40but this gene is apparently not expressed in most adult livers (Guzelian P, Personal communication). One member of the P450III family (P450IIIA5) appears to be expressed in the livers of only one in four adult^.'^,^^ The regulation and catalytic activities of individual P450IIIA subfamily members have not, in general, been well characterized. At least one study suggests that the catalytic activities of these en~ ' inducers zymes may be similar but not i d e n t i ~ a l . The listed in Table 1 appear to increase the aggregate P450IIIA catalytic activity and immunoreactive protein in the adult liver; however, the effects on individual P450IIIA enzymes may differ. It should be noted that, although the drug-metabolizing P450s are generally highly conserved in mam-

''

*Multiple subfamily members have been identified and may be regulated independently. The proteins listed within these subfamilies may therefore not be identical.

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mals, there are significant interspecies differences in regulation of some P450s (reviewed by Gonzalez13). As these differences are defined, it should be easier to identify appropriate animal models when studying human metabolism of specific drugs. For example, gender differences in expression of P450s are common in rats12 but have not been generally observed in human studies. However, evidence has recently been presented that women may have more hepatic P450IIIA activity than do men.39 In summary, multiple P450s are involved in drug metabolism. These enzymes are only remotely related in structure and appear to have evolved from a single gene over 1 billion years ago. The P450 genes are independently regulated and some of the P450s are selectively induced by a variety of commonly prescribed medications.

MOLECULAR BASIS OF P450 INDUCTION The majority of research on the regulation of P450s has to date been performed in laboratory animals. In general, inducers of P450s are also substrates for the P450s they induce. However, this does not always appear to be the case. In most instances, regulation of P450 catalytic activity appears to be at the level of gene transcription.13 After a P450 protein is made in the endoplasmic reticulum, heme is incorporated (presumed in the Golgi apparatus). The role of post-translational modification, such as glycosylation and phosphorylation, of the P450 proteins is poorly understood at present. However, the heme-containing enzyme, or "holoenzyme," probably becomes catalytically active as soon as it is incorporated into the endoplasmic reticulum. Although some studies have suggested that the concentration of P450 apoproteins may exceed that of the holoenzymes in liver micro~ o r n e s , ~there , ' ~ is currently little evidence that incorporation of heme is a regulatable step. In general, the microsomal concentration of the P450 apoprotein (which can be determined immunochemically) correlates well with the catalytic activity characteristic of the ~ 4 5 019.20.22.42.43 . Induction has been best studied in the P450I cytochromes (reviewed by W h i t l ~ c k ~ ~In) .this case, the polycyclic hydrocarbon inducers bind to a cytosolic receptor (aryl hydrocarbon, or "Ah" receptor). The receptor ligand complex then translocates into the hepatocyte nucleus, activating transcription of the P450I genes by mechanisms that have been partially clarified. Induction of P450II and P450III cytochromes also appears to involve transcriptional a c t i ~ a t i o n ,although ~ ~ , ~ ~ the molecular mechanisms involved are currently unknown. In a few situations. induction of P450s involves stabilization of proteins against degradation. The mechanisms involved in degradation of P450 proteins are unknown but the half-lives of individual P450 proteins vary ~ignificantly.~~ In the rat, for example, the half-lives of P450I cytochromes exceed 48 hours48whereas the halflife of P450IIE1 can be less than 10 hours.50 In most cases, induction of P450IIE1 appears to involve a selective prolongation of the half-life of the enzyme without

10, NUMBER 4, 1990

transcriptional activation of the gene.49-51(Stabilization of P450IIE1 mRNA may also be involved in some situation^^^). Induction of P450IIIA cytochromes by macrolide antibiotics appears to involve both accumulation of mRNA and stabilization of the protein^.^' Although it seems likely that stabilization results as a consequence of the drugs binding to these P450s as substrates, other P450s do not appear to be stabilized by their substrate^.^' In animals given a single exposure to an inducer, the increase in catalytic activity of a P450 usually occurs within hours or days after the exposure to inducers (reviewed by Waterman and Estabrook,' Nebert and Gonzalez,'' and Shiraki and G ~ e n g e r i c h ~The ~ ) . P450 activity then returns to baseline within hours or days after removal of the inducer from the body; the actual time taken appears mainly to be a function of the half-life of the P450, which varies depending on the P450 inv01ved.~~ When an inducer is administered chronically to an animal, the time taken to attain a new steady state of an inducible P450 is also a function of the half-life of the enzyme. The available evidence suggests that the situation is similar in man, for example, rifampicin induction of P450IIIA activity can be detected in patients within 48 hours after the initial dose, and it appears that P450IIIA activity will return to baseline within 1 week of stopping the Although little is currently known about the molecular mechanisms involved in induction of human liver P450s, the available evidence suggests that they may be similar to those described in animals. The length of treatment required to produce significant enzyme induction in patients and the time taken to return to baseline activity after stopping a drug will likely vary, depending on the individual P450 involved.

HOW P450s METABOLIZE DRUGS The sequence of events involved in P450 catalyzed metabolism of drugs has been recently reviewed in depth.55 Molecular oxygen binds to the heme moiety of the P450 when the heme iron is in the reduced state. Reduction of the heme iron (Fe+3 to Fef 2, is then accomplished by transfer of reducing equivalents from NADPH. This requires a second microsomal enzyme, NADPH cytochrome P450 reductase. It appears that there is a single form of NADPH reductase that is shared by all microsomal P450s. Drugs bind to the P450 protein in a region (the substrate binding site) that is in close proximity to the heme group. The heme iron is then oxidized (to Fet') and in the process one atom of the molecular oxygen is inserted into the drug, (usually in the form of a hydroxyl group) and the other oxygen atom is reduced though formation of water. The fact that oxidation and reduction occur simultaneously resulted in the older term "mixed function oxidases," which is essentially another term for P450s. The metabolites that result from the catalytic activities of P450s may be less bioactive than the parent drug. On the other hand, the insertion of an oxygen atom often results in a more reactive molecule that can be toxic or carcinogenic in some circumstances.

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It was initially believed that each of the P450s involved in drug metabolism had extremely broad "substrate specificities," that is, any individual P450 was capable of metabolizing most drugs to some degree. This is because multiple P450s are usually capable of metabolizing any given drug when a high concentration of the drug is presented to liver microsomes or to reconstituted systems containing purified P450s. It has recently become clear, however, that at the concentrations attained in the livers of treated patients, many drugs may be largely dependent on single forms of P450 for their metabolism. This is because the relative binding affinities for specific drugs vary greatly among the various P450s. The concept of catalytic specificity is schematically illustrated in Figure 2. After a lypophilic drug enters the hepatocyte, it arrives at the endoplasmic reticulum either by passive diffusion or active processes that have yet to be defined. The many different forms of P450 are attached to and embedded in the endoplasmic reticulum. Each P450 protein has a unique enzyme active site capable of binding some but not all drugs. For example, drug A is capable of binding to and being metabolized by P450I because it "fits" into the substrate binding site (Fig. 2). Drug A will not bind P450II or P45011I. On the other hand, drug B has the appropriate structure to bind to P450II but will not bind to P450I or P450III. However, it is clear that each individual form of P450 has considerable latitude in the various drug structures it is capable of binding, so that any one form of P450 probably is capable of metabolizing many drugs. This must be so, since literally thousands of drugs, xenobiotics, and endogenous compounds are handled by a system that is unlikely to contain more than 50 to 100 different enzymes. This is shown schematically in Figure 2 by drugs C and D. Each drug has a unique structure but each "fits" into the substrate binding site of P4501II. Finally,

some drugs are capable of being acted on by multiple P450s that may not be within the same gene family. This presumably results from a drug having multiple distinct regions of its structure that are capable of binding P450s (not shown in Figure 2). It should be pointed out that, although the concept illustrated in Figure 2 is useful, it has so far been generally difficult to discern common structural elements in drugs metabolized by a single P450. As a result, it is currently difficult to predict which P450 will metabolize a given drug based solely on its structure. Investigators have therefore used several approaches to match metabolic pathways with specific P450s. These have included attempts to block metabolism of a drug in human liver microsomes by P450-specific antibodies,19~39~42~43~5ddho as well as assaying the catalytic activities of purified P450s19.21,59-68 and of enzymes produced by cells transfected with human P450 C D N A S . ~ ' , ~Table ~ - ' ~ 2 gives a partial listing of drugs currently believed to be largely metabolized by human P450s that are members of a single gene subfamily. Table 2 points out that P450s commonly catalyze a variety of reactions in addition to hydroxylations. This is because insertion of molecular oxygen into drugs at TABLE 2.

Reuction type cutalyzedf IA2

IIC*

IID*

IlEl IIA*

FIG. 2. Catalytic specificity of P450s. In order to be metabolized, drugs must bind to the P450 at the "substrate binding site," which is in close proximity to the enzyme's heme prosthetic group. The binding affinity between individual drugs and each P450 may vary significantly so that, at blood concentrations normally attained in patients, a single P450 may be largely responsible for the in vivo metabolism of some drugs. Drugs A and B are metabolized by P4501 and P45011, respectively; these drugs will not bind the other P450s shown. However, each P450 appears to be capable of metabolizing multiple drugs. This is shown by drugs C and D, which are both capable of binding a single P450 (P450111).

Some Drugs that Appear to be Substrates for Specific P450s

Phena~etin".".~' Caffeine29.bl.161 TheophyllineZ916' Acetaminophen"" Mephenyt~in'~ Hex~barbital~'.'~' Diazepamlh" Tolbutamide" Sulfinpyra~one'~~ Phenylbutazone"" S~lfaphenazole"~ Oxyphena~ole~~~ Debrisoquin4j BufuralolM Dextromethorphan"' MetoproI01~~~ Other P blockersx9 Perhexilinelz7 Amitriptyline17' Other n e u r o l e ~ t i c s ~ ~ EncainideI7' CodeineIo9 Acetaminophen"" Ethanol6' Erythr~mycinl~,'~ Triacetyloleandomycin (TAO)19'x Nifedipine2"" Cyclo~porine~~.~~.~~

0-deethylation N-demethylation N-demethylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation Hydroxylation

Hydroxylation Hydroxylation 0-demethylation Hydroxylation Demethylation

N-demethylation N-demethylation Ring oxidation Hydroxylations and N-demethylation 6P-hydroxylation 2- and 4hydroxylation Hydroxylations

*Multiple subfamily members exist that may have differing catalytic properties. tif known.

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certain parts in their structure may create unstable compounds that rapidly degrade. For example, insertion of molecular oxygen onto the carbon atom of an N-methyl group results in cleavage of formaldehyde and a net Ndemethylation reaction. Furthermore, a single P450 can catalyze several different types of reactions at different locations on the same drug. For example, P450IIIA catalyzes two hydroxylations and an N-demethylation on three separate locations of the cyclosporine A molecule. An individual P450 may therefore be capable of catalyzing any one of multiple reactions, depending on which drug, or which part of its structure, binds to the active site. The terms "liver demethylases" or the "liver hydroxylases" are therefore inaccurate because they may reflect the catalytic properties of identical P450s. It should also be pointed out that P450s cannot be categorized as being inherently "bad" or "good" in terms of health. This idea came from the observation that P450s are occasionally responsible for generating toxic, mutagenic, or carcinogenic metabolites from more innocuous substances. This has been best described with the P450I cytochromes, which clearly activate many procarcinogens, such as the arylamine~.".'"~~ It has even been postulated that the catalytic activities of P450I cytochromes tend to result in adverse health outcomes because these enzymes may insert the oxygen atom into substrates at positions sterically hindered from the phase I1 enzymes." Any reactive metabolites produced by P450I cytochromes may therefore be especially resistant to phase I1 detoxification. However, it is now clear that production of harmful metabolites is not limited to the P4501 family. For example, P450IIIA cytochromes are capable of activating aflatoxins and other mycotoxins to %nd also appears carcinogenic m e t a b ~ l i t e s ~ ~ ~ ~P450IIE1 to bioactivate some nitrosamines.'' When rats are treated selectively to induce P450IIIA, P450IIE1, or P450I enzymes, they appear generally to be more susceptible to cancers caused by the compounds bioactivated by the induced enzymes. Nonetheless, this induction may simultaneously protect the animal from the toxicities of some other compounds. For example, rats pretreated with "catatoxic" steroids that are potent inducers of P450III enzymes are protected from the toxicities of many drugs7' and may have increased resistance to dimethylnitrosamine-induced liver cancers.77In addition, rats pretreated with 3-methylcholanthrene appear to be significantly protected from the carcinogenicity of aminoazo dyes.7x Indeed, the latter observation initiated the entire field of phase I metabolism research over 40 years ago. In summary, individual P450s simply attempt to act on whatever substance they bind. An adverse health outcome appears to be a function of both the substrate and the specific P450 involved.

INTERPATIENT DIFFERENCES IN CATALYTIC ACTIVITIES OF THE P450 Large interpatient differences in the abilities to metabolize some drugs have been known for decades. It has been known, for example, that the ability to acetylate certain drugs is deficient in a large portion of the popul a t i ~ n . ~ v hisi sdue to an inherited defect in an hepatic

10, NUMBER 4 , 1990

non-P450 enzyme (reviewed by Clarkxn).A relatively recent finding is that there are also large interpatient differences in the ability to perform P450 metabolism on some drugs. This heterogeneity appears to result largely from genetic factors. In the early 1970s it was simultaneously noted that administration of the antihypertensive medication d e b r i ~ o q u i n ~ 'or . ~ 'the antiarrhythmic medication sparteinex' resulted in exaggerated physiologic responses in approximately 10% of all patients who received the drugs. When these patients were studied, it was discovered that they had greatly reduced ability to form hydroxylated metabolites from these drugs. It was possible to type patients conveniently as rapid or poor metabolizers by giving them a single oral tablet of either drug in the evening and collecting urine the following morning;84 the ratio of the hydroxylated metabolites to the parent compounds in the urine clearly distinguished rapid and poor metabolizers. Administration of these noninvasive tests to relatives of patients shown to be poor metabolizers of either drug demonstrated that the poor metabolizer phenotype wasinherited as an autosomal recessive trait. These studies also showed that deficiency in ability to metabolize sparteine and debrisoquin reflected the identical genetic trait.x4The prevalence of the poor metabolizerphenotype has since been shown to vary widely among different ethnic populations, xs-x7 As can be seen from Table 2, the major form of P450 that catalyzes debrisoquin hydroxylation is in the P450IID subfamily. The inability to metabolize debrisoquin has been recently shown to result from defects in the implicated P4501ID gene. The poor metabolizer phenotype can result from at least three separate genetic defect; (apparently involving noncoding portions of the P450IID gene) resulting in defective RNA splicing.RX The aberrant mRNAs are believed to be translated into proteins that are rapidly degraded and are probably not catalytically active." Inherited defects in the ability to perform phase I liver metabolism of other drugs have also been described." For example, the ability to hydroxylate mephenytoin is deficient in up to 25% of the populationgn and, as suggested by Table 2, this appears to result from an inherited defect in P-450IIC genes."-9' This poor metabolizer phenotype also appears to be inherited as an autosomal recessive trait; however, the molecular basis for the defect is not yet known. Deficiencies in the ability to perform P450-catalyzed metabolism of other medications usually appear to cosegregate with the debrisoquin or mephenytoin poor metabolizer phenotypes, but some do not." It therefore appears that inherited defects exist in many different P450s. In addition to genetic factors, nongenetic factors probably also contribute to interpatient differences in the activity of individual P450s. This is suggested by the fact that the frequency distribution of the liver activity of some human P450s in the population does not appear to be bi- or trimodal (the e x ~ e c t e ddistribution of traits under monogenic control). ' For example, although some early data suggested that a subgroup of poor P450IIIA metabolizers might exist,94 it now appears that the distribution of P450IIIA enzyme activity is roughly uni-

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modal in the population.14 Likewise, the distribution of P450IA2 activity, which can vary more than 30-fold among patients,23-" also appears to be roughly unimodal. Since some P450s are inducible by drugs (Table l), it seems likely that factors such as diet or exposures to ubiquitous environmental toxins (such as polybrominated biphenyls [PBBs] and polychlorinated biphenyls [PCBs]) may significantly contribute to the heterogeneous expression of P450s. There are, then, large interpatient differences in the liver content and catalytic activity of many and possibly all of the P450s, reflecting genetic and probably also nongenetic factors. It may therefore be appropriate to think of each patient as having a unique liver "P450 fingerprint" or "P450 profile," which may not remain constant over time.

WHY DO P450s EXIST AND WHY IS THERE SO MUCH INTERPATIENT HETEROGENEITY? P450s have been identified in all eukaryotes examined and many prokaryotic organisms, suggesting that they play a vital role in life. Such a critical role for P450s involved in metabolism of endogenous substances is not hard to imagine. Those enzymes involved in sterol metabolism, for example, might be essential in the synthesis and maintenance of membranes even in primitive, single cell organisms. The origin of P450s involved in drug metabolism is less apparent; they certainly did not evolve to deal with medications. It is now widely believed that these P450s appeared at more advanced stages of evolution to provide protection from potential . ~ ~ are, in fact, thousands toxins in the e n ~ i r o n m e n tThere of potentially toxic compounds produced naturally by plants. In many cases, these chemicals are not metabolically essential to the plant but are adaptive simply because they are toxic, and hence render the plant inedible. In an environment where such plants represent a major potential food source, insect and animal P450s may have evolved to render the plants edible. In a seasonal climate, or in other situations where food sources vary, the ability to induce detoxifying P450s as needed would be a distinct advantage. For example, when certain insect larvae eat a leaf containing a natural toxin, they rapidly develop the ability to inactivate that toxin by inducing a P450.9hInduction of P450s in insects appears to explain the rapid development of resistance to some commercial pesticides. For this reason, inhibitors of insect P450s are commonly added to pesticides. The fact that human P450s metabolize so many drugs is not coincidence. Most drugs in use today are derivatives of chemicals that are naturally present in our environment. Moreover, the general requirements that oral medications have lipophilicity and have a manageable tissue half-life (excreted within hours of dosage administration) essentially define substrates for the P450 system. In other words, the properties of drugs that are essential for their clinical practicality often make them ideal substrates for the P450s. The importance of the drug-metabolizing P450s to the health of normal human beings is controversial (re-

viewed by Gonzalez et all6). It would seem logical that P450 forms with high frequencies of poor metabolizer phenotypes are not essential for life. The multiple defective alleles for P450IID, for example, may signify propagation of random mutations with no adaptive consequence. However, as previously discussed, the catalytic activity of an individual P450 may be both beneficial or detrimental, depending on the substrate it encounters. It is therefore possible that selective pressures favor either the poor or rapid metabolizer phenotypes, depending on the environmental influences impinging on a particular population of patients. For example, some evidence suggests that rapid metabolizers of debrisoquin are ~"~ poor metabmore prone to lung ~ a n c e r , ~ ' - whereas olizers may be at increased risk of early onset of Parkinson's disease.lo2 An intriguing observation in this regard is that a compound that can cause a Parkinsonlike illness (7-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine [MPTP])'03 appears to be metabolized by P45011D.104It is therefore possible that the health consequences to an individual of exposure to the environment depend on both the xenobiotics present and his (her) P450 profile. This may explain why the prevalence of the debrisoquin poor metabolizer phenotype varies dramatically in different regions of the world.

CLINICAL SIGNIFICANCE OF P450s Drug Actions and Reactions Physicians have long accepted the fact that to achieve a desired therapeutic effect, some patients often require more of a given medication than others. Indeed, physicians are generally advised to "start low and take it slow" when prescribing any medication with a narrow therapeutic plasma concentration range or significant physiologic effects. The discovery of interpatient heterogeniety in the P450s provided a plausible molecular basis for this phenomenon. There are several ways the activity of a single P450 could determine an individual's response to a drug. If the therapeutic effect of the drug correlated with the blood levels of the parent compound, the optimal dose of drug might be greater in patients with high activity of one or more of P450s involved in metabolizing the drug. However, in order for the activity of a P450 to determine the blood levels of a drug, the P450 activity must be rate limiting in the elimination of that drug. This cannot be assumed because many drugs have multiple pathways for metabolism and elimination. Moreover, the elimination of a lipophilic drug involves many sequential steps, as illustrated in Figure 1. The drug must first be delivered to the liver in blood, taken up from the circulation by the hepatocyte, and transported to the endoplasmic reticulum. The drug must then "find" and bind to its appropriate P450. The metabolite or metabolites produced must often then be sorted to phase I1 enzymes prior to being excreted from the hepatocyte. Nonetheless, there are now at least several important examples in which the relationship between drug dose, blood levels, and therapeutic response in an individual patient is largely determined by the catalytic activity of P450IID (the debriso-

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quin hydroxylase). For example, patients with the debrisoquin poor metabolizer phenotype demonstrate roughly threefold and sixfold increases in the area under the plasma concentration time curve for debrisoquinR2 ' ~ ~a result, poor metabolizers generand r n e t o p r ~ l o l . As ally attain therapeutic effects from these drugs at significantly reduced daily dose^.^^,'^^ Poor metabolizers of debrisoquin also generally require lower doses of some tricyclic antidepressants than do extensive metabolizers to achieve therapeutic effects (reviewed by Brosen and Gram'"). Another example of a P450 whose activity appears to be rate limiting in the elimination of drugs is P450IIIA. The daily dose of cyclosporine required to attain a target blood levels can vary up to tenfold among patients receiving the drug.'07 P450IIIA is the major liver ~ ~ , ~as~ previenzyme that metabolizes c y c l o ~ p o r i n eand, ously discussed, the liver catalytic activity of P450IIIA enzymes can vary at least tenfold among patients. We have recently used the erythromycin breath test39 (Table 3) to measure P450IIIA catalytic activity in 32 patients with psoriasis scheduled to receive treatment with cyclosporine for 16 weeks.lo8 We found that the mean trough blood levels of cyclosporine correlated inversely with the patients' P450IIIA activity. This strongly suggests that P450IIIA activity is rate limiting in the elimination of cyclosporine. Moreover, we found that in 27 of the 32 patients studied, the observed mean blood level of cyclosporine was accurately predicted (within 50 ngiml) by a simple linear equation incorporating the patient's daily dose, breath test result, and age.''* This represents the first proposed use of a selective and noninvasive test of the activity of a P450 to estimate appropriate dosing for all patients receiving a drug, and not just a subset of "poor metabolizers. " The activitv of a P450 could also determine an individual's response to a drug if a metabolite produced by a P450, and not the parent compound, was the active therapeutic agent. An example of this may be codeine, which is converted to morphine in the liver by P45011D.10' Poor metabolizers of debrisoquin may therefore have reduced analgesic effects from standard doses of codeine. In summary, it is clear that the profile of P450s in an individual's liver can be a primary determinant of the response to drug therapy. Although the study of human P450s has really just begun, it has already significantly improved our understanding of some well-known clinical observations. TABLE 3. Some Noninvasive Tests that May Assay Activity of Specific P450s P4501A2 P45011C P450IID P45011E1 P450IIIA

Caffeine breath test" Urine caffeine metabolite ratio"' Urine mephenytoin metabolite ratio"' Urine debrisoquin metabolite ratiox' Urine dextromethorphan metabolite ratio1'' N-dimethyl nitrosamine (NMDA) breath test*'"" Erythromycin breath test" Nifedipine urinary metabolite ratios"." Urinary 6 P cortisol:free cortisol (6 PFIFF)" Estrogen 2-hydroxylase radiometric assay1"'

*Not suitable for patient use, since NDMA is a known carcinogen.

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Drug Interactions Because the catalytic activity of P450s may be rate limiting in the elimination of many drugs, it should not be surprising that many clinically important interactions between drugs result from inhibition of these enzymes. Inhibition of P450s can occur by several mechanisms (reviewed by Ortiz de Montellano and Reich'lO)and only a few clinically relevant examples will be discussed here. Drugs that require metabolism by the identical P450 should compete for binding to and metabolism by that P450 (drugs C and D in Figure 2). In theory, any two drugs that are metabolized by the identical P450 (Table 2) have a potential for this form of interaction, but the clinical significance of the interaction will rely on the drugs' relative affinities for binding to the P450, concentrations achieved in the endoplasmic reticulum after therapeutic doses, dependence on the P450 for elimination, and therapeutic ratios. A good example of such an interaction appears to be the elevation of cyclosporine blood levels, and often cyclosporine toxicity, seen when patients receiving cyclosporine are started on treatment with erythromycin."' In this situation, both drugs are competing for binding to, and metabolism by, P450IIIA in the liver, and also possibly the gut (see "Role of Extrahepatic P450s"). The inhibition of cyclosporine metabolism by erythromycin should be proportional to the concentration of erythromycin present in the endoplasmic reticulum and should be completely reversible on discontinuing the erythromycin. (Erythromycin may also inhibit P450IIIA by noncompetitive mechanism^,"^ see discussion later). Reported interactions between cyclosporine, calcium channel blocker^,^^'-"^ and ketoc ~ n a z o l e "are ~ presumably also due, at least in part, to competitive inhibition of P450IIIA activity.28 The "steroid sparing" effect of triacetyloleandomycin (TAO) in steroid-dependent asthmatics has been shown to result from reduced steroid metabolism (reviewed by Descotes et al1I7)and this probably also results from P450IIIA inhibition by TAO. Examination of Table 2 suggests potential interactions that have not been investigated. For example, the metabolism of calcium channel blockers should be reduced in patients receiving cyclosporine, and this may be why these drugs are particularly efficacious in treating hypertension in these patients. It should be pointed out that some drugs appear to bind to, and act as inhibitors of P450s that do not appear to actually metabolize the drug. For example, quinidine selectively inhibits P450IID in human liver microsomes and in patients in vivo. After a single oral dose of quinidine, extensive metabolizers of debrisoquin will transiently eliminate the P450IID substrates metoprolol or encainide (Table 2) as if they had the poor metabolizer p h e n ~ t y p e . " ~ , Nonetheless, "~ quinidine appears to be metabolized mainly by P450IIIA and not P45011D.57 In addition to competitive interactions with P450 substrate binding sites, some drugs appear to interact directly with the heme iron, resulting in inhibited catalytic activity (reviewed by Ortiz de Montellano and ReichllO). In studies in microsomes, cimetidine and other H, antagonists appear to inhibit one or more P450s on this ba-

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sis.I2' However, the drug concentrations needed to inhibit metabolism in microsomes (millimolar range) usually far exceed blood levels attained in patients receiving the drugs. Moreover, some H, blockers that are potent inhibitors of drug metabolism in microsomes do not appear to cause much inhibition in vivo.lZ0Thus, the mechanism whereby some H, antagonists inhibit the P450-mediated metabolism of other drugs in vivo remains unclear. Finally, some drugs appear to be converted by P450s to metabolites that then bind to the P450 protein or heme group, noncompetitively abolishing catalytic activity. In patients treated with 8-methoxypsoralen for psoriasis, for example, the ability to metabolize and eliminate caffeine, is dramatically reduced."' This appears to be due to binding of a psoralen metabolite to a subset of P450 proteins, presumably including P450IAl (Table 2) resulting in their irreversible, or "suicide" inactivation.lZ2Some macrolide antibiotics are converted to reactive metabolites that tightly and selectively bind to the heme moiety of human P4501IIA cytochromes, also resulting in inactivation.19 In addition to the various mechanisms resulting in inhibition of P450 catalytic activity, drug interactions can involve induction of P450s. Comparisons of inducers and substrates for individual P450s (Tables 1 and 2, respectively) indicate possible interactions on this basis. For example, rifampicin treatment results in significant induction of P450IIIA (Table 1) and this presumably is the basis for reports of increased metabolism of cyclosporine and prednisone (P450IIIA substrates, Table 2) in The fall in cyclosporine patients receiving rifampin.'23.124 blood levels seen in patients treated with phenytoini2* and carbamazepine12' can presumably also be explained by induction of P450111A.28The failure of birth control pills in women receiving rifampin may also be due to induction of P450IIIA. l 4 In summary, because the catalytic activity of at least some P450s appears to be rate limiting in the elimination of many drugs, interactions are possible whenever a patient concomittantly receives two drugs that bind to the same P450. When patients receive inducers of a P450, they may have accelerated clearance of any drug that is metabolized by that P450. As the lists of drug substrates and inducers for each P450 are determined in the future, the molecular basis for many more recognized drug interactions should become evident. Furthermore, it should in the future be possible to avoid potential interactions before they are observed.

Hepatotoxicity Many drugs and other compounds are toxic to the liver when ingested in large doses. Some drugs can also cause significant liver injury in a minority of patients who receive them at doses that are normally considered therapeutic. This unpredictable or "idiosyncratic" toxicity may result from an immune-mediated, or "allergic," response to a drug. However, it is likely that interpatient differences in the catalytic activities of P450s account for much, if not most, idiosyncratic hepatotoxicity. A patient's P450 profile could predispose him (her)

to hepatotoxicity in at least two ways. First, if the drug itself is hepatotoxic, reduced ability to detoxify or eliminate the drug will predispose the patient to toxicity. For example, liver and neurotoxicity caused by chronic treatment-with perhexiline appears to be more common in patients with the debrisoquin poor metabolizer phenotype.',' P450IID (Table 2) presumably catalyzes the usual major pathway for detoxification and elimination of perhexiline and the drug therefore accumulates in poor metabolizers. The second and a more usual basis for drug hepatotoxicity is when relatively innocuous drugs are converted by P450s into reactive and potentially toxic metabolites. The best-studied e x a m ~ l eof this is acetaminophen toxicity. Acetaminophen, which already contains a hydroxyl group, does not require phase I oxidation in order to undergo phase I1 conjugation to glucuronide or sulfate. As a result, less than 5% of administered acetaminophen is normally metabolized by P450s; however, it is this pathway that appears to produce the electrophilic metabolite or metabolites that are toxic to the hepatocyte.12' A major P450 involved in production of electrophilic metabolites from acetaminophen appears to be P450IIEl. '29.130 After a therapeutic dose of the drug, hepatocellular injury is normally averted because the electrophilic metabolite or metabolites are efficiently detoxified by conjugation to glutathione. However, when a large dose of acetaminophen is consumed (usually greater than 15 gml"), the normal phase I1 pathway becomes saturated and a large amount of the drug undergoes P450 catalyzed oxidation to the toxic metabolite or metabolites. This exhausts the available glutathione stores, resulting in intracellular accumulatio~of the toxic metabolites, and hepatocellular injury. It has recently been appreciated that chronic alcoholics can develop acute acetaminophen toxicity after ingesting far less than 15 gm of the drug.',' TWOcomplimentary theories have been proposed to explain this enhanced susceptibility to acetaminophen toxicity. The first theory is that the livers of alcoholics are depleted in glutathione and are therefore incapable of detoxifying the metabolites produced by P450IIE1 and perhaps other P450s. The second theory is that alcoholics actually produce more of the toxic metabolite at any given dose of acetaminophen because P450IIE1 is induced by ethanol (Table 1). If induction of P450IIEl alone increases susceptibility to acetaminophen toxicity, we would predict that patients treated with the P450IIE1 inducer isoniazid (Table 1) should also be more susceptible. We have, in fact, recently described a patient who, while receiving isoniazid, developed unexpectedly severe liver and renal injury after ingesting acetaminophen.I3' Because isoniazid metabolism in the liver does not appear to involve generation of e l e ~ t r o p h i l e s , it' ~is ~ unlikely that this patient's glutathione stores were diminished. This case therefore suggests that induction of P4501IE1 alone may predispose patients to idiosyncratic acetaminophen toxicity. It should be noted that the acute administration of ethanol actually protects laboratory animals from acetaminophen t 0 x i ~ i t y . lThis ~ ~ effect is expected, since ethanol is substrate for P450IIE1 (Table 2) and should therefore compete with acetaminophen for binding to

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(and metabolism by) P450IIE1 (as demonstrated by drugs C and D in Figure 2). Competitive inhibition of P450IIE 1 by ethanol may therefore counteract the effect of induction of P450IIEI in the inebriated alcoholic patient. (It has recently been suggested that the ingestion of ethanol may result in reduction in the supply of NADPH in the endoplasmic reticulum and that this may also explain the fall in P450 catalyzed metabolism of acetaminophen in the presence of ethan01.l~~ However, such an effect should decrease the catalytic activity of all P450s and in rats ethanol does not appear to influence the in vivo catalytic activity of P450III enzymes"' significantly.) In theory, intravenous ethanol might therefore be an appropriate adjunct to N-acetyl cysteine therapy when treating alcoholics who have recently taken an overdose of acetaminophen. However, this cannot be recommended, since ethanol is a hepatoxin and since administration of N-acetyl cysteine alone is a highly effective therapy. As previously discussed, P450IIE1 appears to have a relatively short half-life, at least in rats. The activity of P450IIE1 toward acetaminophen may therefore remain at induced levels for only hours after the ethanol is eliminated from the body. This may explain why in one study, alcoholics given acetaminophen did not appear to produce more electrophilic metabolites than did nonalcoholic~. It has been reported that laboratory animals are protected from acetaminophen toxicity by pretreatment with ~ i m e t i d i n e ,and, ' ~ ~ largely on this basis, it has been suggested that cimetidine may have a role in the treatment of acetaminophen overdose."' A key question, then, is whether P450IIE1 is inhibited by cimetidine in alcoholics. Purified human P450IIE1 does not appear to be inhibited by cimetidine in a reconstituted system (Raucy J: Personal communication). Studies that have claimed a therapeutic benefit from cimetidine in acetaminophen toxicity have generally used animals that were pretreated with inducers of P450IA2 (3-methylcholanthrene) or P450IIB1 (phenobarbital). It is likely that P450IIE1 makes a minor contribution to the total P450 metabolism of acetaminophen in these animals. Toxic metabolites may be produced from acetaminophen by human liver P450s other than P45011E1, most notably P4501A2."1 Since cimetidine inhibits elimination of the P450IA2 substrate caffeine,"' it seems likely that cimetidine inhibits P450IA2 activity. However, 8methoxypsoralen may be a more effective inhibitor of P450IA2 in patients than is cimetidine. Evidence for this is that caffeine elimination is inhibited to a greater degree by 8-methoxyp~oralenl~~ than by ~ i m e t i d i n e . ' ~8-' Methoxypsoralen may also inhibit P450IIE1 (unpublished observations). Although 8-methoxypsoralen cannot be recommended in the treatment of acetaminophen overdose, it may be appropriate to explore its use in conjunction with N-acetyl cysteine in controlled clinical trials. Finally, it has recently been shown that P4501IE1 is exclusively expressed in zone 3 (pericentral) hepatocytes.:' This may explain why acetaminophen injury is characteristically centrilobular. In summary, then, it seems likely that induction of P450IIE1 by ethanol, and possibly by other drugs, predisposes individuals to acetaminophen toxicity. Alcohol-

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ics appears to be particularly at risk because they may also be depleted in hepatic glutathione stores. In spite of these concerns, acetaminophen may be the safest nonsteroidal anti-inflammatory agent currently available for the alcoholic. 14' As mentioned earlier, some examples of idiosyncratic hepatoxicity appear to involve immunologic mechanisms. A recent finding is that this type of liver injury may also involve P450s in some cases. For example, patients who develop an idiosyncratic hepatitis from the diuretic tienilic acid have a very high incidence of circulating autoantibodies that react with microsomes prepared from liver and kidney. These antibodies are termed antiliver and antikidney microsomes type 2, or antiLKM2.I4' It has recently been discovered that the major antigen recognized by serum from these patients is a P450 protein within the P450IIC subfamily (the mephenytoin hydroxylase) which also appears to be a major P450 involved in the metabolism of tienilic acid.I4' It has been proposed that P450IIC produces a metabolite from tienilic acid that is so reactive that it immediately binds to the cytochrome protein that created it. 14' This appears to result in altered antigenicity of the P450IIC protein, stimulating the production of antibodies that recognize the unaltered ("normal") P450IIC. Halothane metabolism in rats appears also to produce antigenically altered P450s that are members of the P450IIB gene subfami l ~ . Anti-LKM ' ~ ~ antibodies have been observed in patients with liver injury from other drugs,14' and it remains unclear whether the anti-LKM antibodies are an epiphenomenon or, in fact, mediate an immune attack on the liver.144For the latter to be the case, it would logically follow that the P450s be present on the surface of the hepatocyte and studies have produced conflicting results in this regard. 145.'46 A subpopulation of patients with autoimmune chronic active hepatitis also have anti-LKM antibodies. 14' These antibodies, termed "anti-LKM 1 ," have been recently shown to react with P450IID, the debrisoquin hydroxylase (Table 2).148.14' These patients have generally not received medications known to be metabolized by P450IID. A plausible theory is that these antibodies are caused by oxidation of substances present in the environment or in the diet, creating reactive and antigen-altering metabolites. It is currently unknown if this form of autoimmune hepatitis is more common in individuals with the extensive metabolizer phenotype for P450IID. Thus, it is clear that idiosyncratic hepatoxicity can result from either increased activity of individual P450s involved in creating toxic metabolites or from reduced activity of P450s involved in detoxification or elimination. In some cases, antibodies to individual P450 proteins have been found in patients with drug-induced liver disease, and in some patients with autoimmune hepatitis. The role these antibodies play in the etiology of liver injury has not been established.

ROLE OF EXTRAHEPATIC P450s Virtually every tissue in the body contains P450s, although their roles are generally poorly understood at

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Dresent. It has been known for some time that enterocytes are capable of phase I metabolism.liO In most cases the ability of enterocytes (or of various subcellular fractions prepared from enterocytes) to metabolize drugs is small compared with similar measurements made in liver tissue. However, it has recently become clear that the profile of P450s present in human gut mucosa is not identical to that vresent in human liver.15' In an uninduced state, P450IIIA enzymes appear to account for more than 70% of the total P450 present in human jejunal m u ~ o s a ,whereas '~~ they account for less than 20% of the total P450 present in the liver.I9 The microsomal concentration of ~ 4 5 0 1 1 1proteins, ~ however, appears to be comparable in human enterocytes and liver.I5' The P450IIIA genes expressed in human jejunum appear to be identical to those expressed in human liver. 153 The role of intestinal P450IIIA enzymes in drug metabolism is largely unexplored but there is indirect evidence suggesting that it may be important. Drugs known to be substrates for P450IIIA enzymes (Table 2) generally have poor oral bioavailability that may vary significantly among individuals. We have recently shown that P450IIIA enzymes metabolize cyclosporine in rat enterocyte microsomes, and that this activity exceeds that present in liver microsomes prepared from the same animals.Is4 At least in the case of cyclosporine, the available evidence suggests that what has been interpreted as poor oral bioavailability may largely represent metabolism in the enterocyte by P450IIIA enzymes. For example, the interaction between cyclosporine and erythromycin cannot be totally explained by competitive inhibition of P450IIIA activity in the liver.15' In fact, erythromycin appears to increase greatly the "absorption" of cyclosporine.ls5 Competitive inhibition of P450IIIA activity by erythromycin present in the enterocyte might allow more cyclosporine to enter portal blood, accounting for this observation. The importance of intestinal P450IIIA enzymes is also supported by the observation that "absorption" of cyclosporine appears to be significantly reduced in patients receiving the P450IIIA inducers p h e n y t ~ i n lor~ ~rifampin."' We have recently shown that rifampin induces P4501IIA proteins in explant cultures of human jejunal mucosa (unpublished observations), suggesting that regulation of P450IIIA cytochromes may be similar in intestine and liver. Induction of intestinal P450IIIA enzymes is therefore an attractive, but as yet unproven, hypothesis to account for reduced oral bioavailability of cyclosporine in patients receiving inducers of P450IIIA. In summary, P450IIIA cytochromes appear to be particularly prominent in intestinal mucosa where some evidence suggests that they may contribute significantly to the poor oral bioavailability of some drugs. Interactions between drugs and P450s at the level of the intestinal mucosa may be clinically important, although this has been largely unexplored.

FUTURE PROSPECTS FOR INVESTIGATION OF CYTOCHROMES P450 We know the most about the health consequences of the debrisoquin polymorphism simply because non-

invasive means of phenotyping individuals have been available for roughly a decade. In order to study the health consequences of interpatient variation in other as0 appropriate means of selecpects of the ~ 4 5 profile, tively and noninvasively determining the activity of the other P450s will have to be developed. As genetic defects accounting for inherited polymorphisms are defined, genetic analysis of white blood ceils may provide a convenient wav to screen for these defects.''' However, it seems unlikely that genetic studies will obviate the need for noninvasive catalytic assays that are P450 specific. This is because there may be multiple defective alleles for any given P450 in the p o p ~ l a t i o n and , ~ ~ nongenetic factors appear to contribute significantly to interpatient heterogeneity in the activities of at least some P450s (Table 1). An individual's P450 profile could be determined by traditional pharmacokinktic measurements, such as the clearance or terminal rates of blood elimination for model P450 substrates. These studies are generally difficult to perform, however, and probably could not be routinely undertaken in a physician's office. Ideal tests would therefore assav metabolites of model substrates (which might include endogenous hormones) in easily collected bodily excretions, such as breath, urine, or possibly saliva. Table 3 lists some of the recently proposed noninvasive assays that may become a major part of clinical practice in the future. It may be possible to use single drug "probes" that are metabolized by multiple P450s and that may therefore provide information '~~ about multiple P450s s i m u l t a n e ~ u s l y . Alternatively, multiple P450 specific probes could be simultaneously administered to patients in a "cocktail. " I s y The knowledge of relevant aspects of an individual's P450 profile should assist future physicians in individualizing dosing of many drugs. Such knowledge should also make it possible to predict and thereby avoid many drug interactions and adverse reactions such as hepatotoxicity. The day may come when a patient's P450 profile is listed, along with medication allergies, on the front of the medical record or inpatient chart. It should also be possible to use selective inducers and inhibitors of P450s to alter an individual's P450 profile for therapeutic benefit. For example, it has recently been reported that the cost of cyclosporine therapy can be dramatically lowered by administering the drug with ketocona~ole."~ As previously discussed, this is a predictable interaction that appears to result from inhibition of P450IIIA activity by ketoconazole. On the other hand, it may be desirable to increase P4501IIA activity in some patients. We have recently attempted therapeutically to induce P450IIIA in a liver transplant recipient.Is6 Undoubtedly, tests will be developed in the future that will make it possible for a physician to determine at least certain aspects of patients' P450 profiles. In some cases, it should also be possible, through the use of selective inducers and inhibitors, to alter a patient's P450 profile for therapeutic benefit. It has even been predicted that "cytochrome P450 doctors" will create a new medical subspecialty in the future.lhO Finally, the improved understanding of the P450s in man should have dramatic implications for the pharmaceutical industry. It is now possible to transfect yeast or

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stable mammalian cells lines with P450 cDNAs and thereby direct expression of individual, catalytically active proteins. It will soon be possible to determine which human P450s metabolize a given drug simply by adding them to test tubes containing these reconstituted enzymes. Once the spectrum of P450 profiles in the population is known, it will thus be possible to gain valuable information about how a potential drug will be handled by a population of patients before the drug actually reaches clinical trials. This should greatly speed development of potential therapeutic agents while reducing the cost and potential morbidity of initial clinical trials. In addition, if subsets of patients likely to have "idiosyncratic" reactions to a given drug could be identified with suitable noninvasive tests, this should allow the marketing of many potentially useful drugs that would not currently receive FDA approval for clinical use.

SUMMARY The cytochromes P450 may represent the major metabolic frontier between the environment and the body. The recent discovery of interpatient differences in P450 profile has provided a plausible explanation for heterogeneous dosing requirements for some individual drugs. The heterogeneity in P450 profiles appears to also account for some "idiosyncratic" drug reactions, especially hepatotoxicity. An important challenge for the future is the development of safe and noninvasive tests capable of determining patients' P450 profiles. It is likely that such tests will greatly facilitate individualization of dosing of many drugs in the future. In addition, such tests should be useful in identifying patients likely to develop "idiosyncratic" toxicity to specific drugs. Finally, it seems likely that interpatient differences in P450 profile may at least in part explain interpatient differences in susceptibility to environmental diseases. For example, it may in the future be possible to identify individuals with P450 profiles that render them susceptible to leukemia from xenobiotics such as benzene. Such individuals could be advised not to work in an environment containing these chemicals or, alternatively, to take a medication which reduces their risk by appropriately altering their P450 profile. Identification and in vitro characterization of human P450s is now well underway and may be largely completed within the next decade. However, studies addressing the clinical significance of interpatient differences in P450 profile, as well as the genetic and nongenetic factors that underlie this heterogeneity, are just beginning. This should remain a rewarding area of research for many years to come.

REFERENCES I.

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Role of cytochromes P450 in drug metabolism and hepatotoxicity.

The cytochromes P450 may represent the major metabolic frontier between the environment and the body. The recent discovery of interpatient differences...
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